1. Field of the Invention
The present invention relates to the field of heterogeneously integrated sensor arrays, preferably biosensor arrays. In particular, the invention relates to nanomechanical cantilever type biosensors for detecting target-receptor molecule binding, which produce an electrical detection signal upon change in cantilever mass or static deflection. The invention also relates to the manufacture of nanomechanical cantilever biosensor arrays by parallel assembly of many different biomolecule derivatized nanorods onto single silicon or thin-film electronic circuits using “bottom-up” integration strategies.
2. Description of the Prior Art
During the past decade, there: has been an increasing interest from the commercial and government sectors to develop on-chip integrated biosensor arrays that can selectively report the presence and quantity of specific biomolecules contained in sample populations. Examples of such populations range from cells and model organisms for pharmaceutical and genome research to samples of the environment for pathogens and biological warfare agent detection. In the case of genome research, it is necessary to discover gene sequences that provide a blueprint of the cell or organism through systematic identification of known and predicted genes. It is also of great interest to use the so-called genomic arrays for gene expression monitoring and for screening sequence variants or mutations. For these applications, it would be desirable to survey >1 Mbit of genomic information on a single chip that is only a few cm2 in area. In contrast, biosensors used to screen pathogens may have relaxed requirements on the number of different biomolecules that must be sensed simultaneously, while placing greater emphasis on detection time, minimum detection levels, field durability, overall system size, and cost.
Most biosensor platforms are based on the extremely selective recognition principles inherent in biological systems. Bioreceptors that have been used as sensing elements include biomolecules such as antibodies, enzymes, and nucleic acids. When a receptor undergoes a binding event with a target biomolecule, the information collected by the sensing element regarding the receptor-target attachment must be converted into a signal that can be easily measured. There are a number of transduction mechanisms that can be exploited for converting this attachment information, including optical, electrochemical, magnetic, and mass sensitive measurements. The choice of the particular bioreceptor/transducer combination will ultimately impact biosensor figures of merit such as detection sensitivity, selectivity, integrability, scalability, and cost.
Most commercially available integrated biosensors rely on optical sensing of antibody-antigen interactions and complementary DNA hybridization. One example involves fluorescently-tagged target DNA that hybridizes with complementary receptors on the sample. DNA microchip arrays are based on this principle, and have increased rapidly in sophistication and density as a result of a number of recent technological developments. First, non-porous substrates such as glass were introduced, facilitating miniaturization of fluorescence detection. Second, semiconductor photolithography techniques were adapted to control the spatial synthesis of oligonucleotides. Currently, these “gene chips” can contain as many as 400,000 distinct oligonucleotides, where each sensor element occupies ˜20 μm2. These developments have made possible large-scale scientific studies on gene expression (i.e., what the protein product of the gene does) and, to a lesser extent, gene variation (i.e., identifying SNPs). While the progress on “gene chips” has been impressive, continued miniaturization will require significant advances in improving the sensitivity of fluorescent labeling and optical detection techniques. It is also unlikely that extensions to this technique will provide quantitative data regarding the concentration of particular DNA sequences, especially for extremely low concentrations of DNA. Finally, because nonspecific binding of SNPs is unacceptably high even with short oligonucleotides, further refinements are needed before gene variations can be thoroughly studied using these biosensor arrays.
There are several other techniques that also rely on fluorescent tagging and/or optical readout, which have not matured commercially to the level of the “gene chip”. These include surface plasmon resonance, evanescent dielectric waveguides, self-encoded beads, and self-encoded nanoparticles. The first two are capable of collecting kinetic data for hybridization in situ due to their surface selectivity. The last two encode sequence information optically rather than spatially (as in gene chips): polymer beads with fluorophores of distinct wavelengths, and nanometer-scale rods with bars of metals having high contrast. Self-encoded nanoparticles remove many of the limitations associated with transport of target molecules to the sensor elements on planar arrays by suspending the nanoparticles in solution during hybridization and readout. However, each of these techniques still requires sophisticated optical detection, which may limit their utility in certain applications.
A technique that has the potential to enhance the selectivity and sensitivity of DNA microarrays labels oligonucleotide targets with metal nanoparticles rather than fluorophores. In this scheme, a three-component “sandwich assay” is used whereby the target hybridizes with complementary receptors immobilized on the sensor surface and on the nanoparticles. Hybridization is detected by first increasing the size of the nanoparticles through Ag electroless plating and then by optically scanning the sample using a flatbed scanner to detect changes in optical contrast between different sensor elements. This work also demonstrates that nanoparticle labels improve significantly the selectivity of oligonucleotide hybridization with single base pair mismatches.
To overcome drawbacks of optical sensing, several groups have initiated research on biosensors that detect the presence of target biomolecules based on magnetic, electrical, or mass sensitive transduction. The bead array counter biosensor uses DNA-functionalized magnetic nanoparticles as the target probe and complementary DNA-covered magnetoresistive materials as the receptor/transducer. When target and receptor DNA hybridize, the magnetic particles bind to the sensing element and modify the local magnetic field. This change is measured electrically by monitoring the resistance of the element, where the resistance is proportional to the number of hybridized beads on the element. Unlike sensors that use optical readout, electronic functions can be integrated directly on chip with the sensing element.
Mass sensitive biosensors are among the newest and perhaps one of the most promising approaches for applications requiring high-density, on-chip integration of receptor/transducer and signal/data processing functions. It has been demonstrated that large-area quartz crystal microbalances (QCMs), which measure the shift in resonant frequency of a quartz crystal oscillator due to mass changes by target-receptor binding, are up to 100 times more sensitive than DNA microarrays. While it is difficult to scale and integrate QCM technology, two groups have investigated a related approach that uses silicon micromachined cantilevers as strain- or mass-sensitive transducers. Receptor DNA was attached to the top surface of 100×500 μm2 cantilevers and the static deflection of the cantilever due to surface stress induced during DNA hybridization was measured optically. Deflections of 17 nm were induced when large (400 nM) concentrations of complementary target DNA were analyzed.
Additionally, some patent prior art has addressed the need for integrated biosensors. Two published patent documents in the field of micromechanical biosensors are U.S. Pat. No. 6,289,717 to Thundat et. al. and PCT publication WO 98/50773 to Charych et. al. Thundat discloses a sensor apparatus comprising a microcantilevered spring element having a coating of a detector molecule wherein the spring element bends in response to a binding event. Similarly, Charych uses a microfabrication process to produce a thin deposition of piezoelectric material to produce a microcantilever that responds to a binding event with an electrical signal.
Despite prior art teachings there continues to be a need for heterogeneously integrated biosensors having improved properties and advantages. This is the need addressed by the present invention. While prior art practice for integrated mass sensitive biosensing is focused on cantilever structures based on standard silicon micromachining techniques, the present invention describes a nanomechanical cantilever type biosensor that offers improved sensitivity, selectivity, and dynamic range. The present invention also describes methods for producing biosensor arrays based on parallel assembly of many different biomolecule derivatized nanorods onto silicon or thin-film electronic circuits using bottom-up integration strategies.
Accordingly, it is an object of the present invention to provide a new an improved method of making nanomechanical devices.
Another object of the present invention is to provide a method of making nanometer scale transducers using “bottom-up” integration.
Another object of the present invention is to provide a method of making nanomechanical transducers having electrofluidic or fluidic directed assembly.
A further object of the invention is to provide a nanoscale transducers that are useful as chemical or biological sensors.
Yet another object of the invention is to provide nanoscale transducers having nanomechanical cantilevers providing sensitive and selective detection capability.
These and other objects and advantages of the invention and equivalents thereof, are described and provided in the drawings and descriptions that follow and manifest in the appended claims.